- Email: [email protected]
Screening for BRCA1-pathway Inactivation in Sporadic Breast Cancer Cell Lines
Volume 87 Number 2S Supplement 2013 These results have now been investigated on the molecular level in these CHO cells and in human cancer cells deficient in specific proteins of HR. Materials/Methods: Experiments were performed with an HR-proficient and -deficient CHO cell line pair, NHEJ-deficient CHO cells and the corresponding genetically reconstituted wild-type cells, a human ovarian tumor cell pair (BRCA2-deficient and wild-type, respectively) and the mammary carcinoma MDA-MB-436 (BRCA1-deficient) and MDA-MB231 (BRCA1-wildtype) cells. Results: In contrast to NHEJ-deficient cells, HR-defective Irs1sf cells and CHO-wild-type cells, depleted of Rad51, showed an increased RBE10 in comparison to their corresponding wild-type cells (RBE10 of 1.44 and 1.52 vs 1.33, respectively). This difference in the RBE could be reversed when cells were irradiated in plateau phase (RBE10: AA8 Z 1.29; Irs1sf Z 1.27), indicating a critical role of HR. Likewise, human BRCA2-deficient cells were markedly hypersensitive towards proton irradiation. Furthermore, transient inhibition of ATM in MDA-MB-231 cells by the potent ATM-inhibitor KU60019 significantly sensitized them to proton- vs photon irradiation. Quantification of residual chromosomal aberrations in wildtype and HR-deficient cells revealed a slightly enhanced total amount of chromosomal aberrations in the HR-deficient cells in response to both types of IR. Proton-irradiation resulted in more fragments but also more complex chromosomal aberrations, especially in the HR-deficient cells, with a shift from chromosome- to more chromatide-type aberrations in HR-deficient cells. Conclusions: Our data demonstrate an enhanced susceptibility of HRdeficient cells to proton-irradiation. The enhanced RBE is not due to an increased amount of induced DNA DSBs, but might be linked to the slower repair kinetics in HR-defective cells due to a differential quality of DNA damage induced by proton- versus photon-irradiation. This might become relevant for clinical stratification of patients carrying mutations in the DNA damage response pathways. Author Disclosure: A.O. Fontana: None. N. Grosse: None. E.M. Hug: None. A. Sartori: None. T. Lomax: None. M.N. Pruschy: None.
3134 A Mitotic Pathway for Radiation-Induced Genome Damage S. Bakhoum,1,2 L. Kabeche,1 M. Wood,3 A. Suriawinata,1 R. Louie,3 D. Chan,3 C. Petritsch,3 J. Murnane,3 D. Compton,1 and B. Zaki1,2; 1Geisel School of Medicine at Dartmouth, Hanover, NH, 2Norris-Cotton Cancer Center, Lebanon, NH, 3University of California San Francisco, San Francisco, CA Purpose/Objective(s): The exquisite sensitivity of mitotic cancer cells to ionizing radiation underlies the rationale for fractionated radiation therapy. Nonetheless, the mechanism of this cell cycle-dependent vulnerability is unknown. Materials/Methods: Here we use live cell imaging and high-resolution microscopy to examine the effect of radiation on chromosome segregation and genome damage during mitosis. We also investigate the role of chromosome segregation errors on the viability of irradiated cells using clonogenic survival assays, an orthotopic mouse model of glioblastoma multiforme, and a retrospective clinical study in patients diagnosed with rectal adenocarcinoma. Results: We show that ionizing radiation selectively increases the stability of microtubule attachments to chromosomes at kinetochores, eliciting a dose-dependent surge of kinetochore-microtubule attachment errors during chromosome segregation. These errors, manifested by lagging chromosomes in anaphase, generate whole-chromosome mis-segregation, long-term aneuploidy, a preponderance of micronuclei, and chromosome pulverization leading to chromothripsis. Destabilizing microtubule attachments to chromosomes leads to reduction of these defects, substantial increase in the viability of irradiated mitotic cells (by up to 500-fold), and radiation resistance in orthotopically transplanted human glioblastoma multiforme tumors. Yet, pharmacologically increasing kinetochore-microtubule attachment errors potentiates radiation-induced genome damage. Consistently, we also show that rates of lagging chromosomes predict response to chemoradiation therapy in patients with
Poster Viewing Abstracts S637 rectal adenocarcinoma whereby patients with elevated chromosome missegregation rates exhibit a superior pathological response (OR Z 4.21). Conclusions: Collectively, our data identify a novel mitotic pathway for widespread genome damage beyond direct DNA breaks, and they suggest that kinetochore-microtubules can be usurped to generate this damage for the purpose of tumor radiosensitization. Author Disclosure: S. Bakhoum: None. L. Kabeche: None. M. Wood: None. A. Suriawinata: None. R. Louie: None. D. Chan: None. C. Petritsch: None. J. Murnane: None. D. Compton: None. B. Zaki: None.
3135 HIF-1alpha Posttranscriptional mRNA Regulation Under Normoxia: The Significance of UTRs T. Nishioka, M. Yasuda, and H. Shirato; Hokkaido University, Sapporo, Japan Purpose/Objective(s): HIF-1alpha is a central regulator of cancer progression. HIF-1alpha protein is rapidly degraded at normal oxygen level. Under hypoxia, it escapes from degradation and reaches to a detectable level. In the present study, we investigated: 1) the relation between HIF-1alpha and radio-sensitivity, 2) whether or not HIF-1alpha degradation is due to VHL, and 3) the effect of HIF-1alpha’s 5’- and 3’UTR on translation. Materials/Methods: 1) Colony assay was performed on normal rat fibroblasts that were transfected with HIF-1alpha at a dose 4, 8, and 10 Gy. 2) VHL: Western blotting was carried out on several malignant cell lines expressing different amount of endogenous HIF-1alpha. 3) Luciferase reporter assay: a series of HIF-1alpha constructs containing coding region (C) with or without UTRs were made and introduced into HeLa, H1299, HEK293, B16 F0, B16F10, U87MG, PPC-C2A, MCF7, and MDA-MB453 to measure HRE-Luciferase activity in each cell line. RT-PCR and immunoblotting were also performed at the same experimental conditions. Results: 1) Cells transfected with HIF-1alpha expressed significantly higher survival at each dose (p < 0.05). 2) The VHL level did not always correspond with the expression of HIF-1alpha. 3) For each cell line, deletion of 5’UTR showed a decreased HIF-1alpha expression and deletion of 3’UTR enhanced the expression. A synergetic effect of more than 2-fold was observed for deletion (i.e., 5’ and 3’) for each cell lines. In particular, a highly-aggressive breast cancer cell line MDA-MB-453 demonstrated a 3.8-fold higher transactivational activity compared to a less-aggressive breast cancer cell line MCF7. As to the deletion 5’UTR mutants, a 15- to 20-fold increased transactivational activity was observed for 5’UTR containing constructs compared to the coding region only. RT-PCR revealed that all of HIF-1 alpha constructs expressed the same amount of mRNA. Surprisingly, the cells transfected with 5’UTR containing HIF-1alpha expressed 10 times more amount of proteins compared with coding region only clone. Conclusions: 1) HIF-1alpha increases radio-resistance. 2) In some cells, higher level of HIF-1alpha expression is not due to the expression level of VHL. 3) HIF-1alpha UTRs affects its expression. HIF-1alpha 5’ UTR has an effect of increased protein expression while 3’UTR reduces the protein level. The extent of UTR effect on translation might explain aggressive nature as seen in the breast cancer cell lines. If detailed mechanisms of UTRs on HIF-1alpha translation are clarified, a new path is expected to radiation therapy. Author Disclosure: T. Nishioka: None. M. Yasuda: None. H. Shirato: None.
3136 Screening for BRCA1-pathway Inactivation in Sporadic Breast Cancer Cell Lines M. Thompson,1,2 R. Mutter,3 and S. Powell1; 1Memorial Sloan-Kettering Cancer Center, New York, NY, 2University of Pittsburgh School of Medicine, Pittsburgh, PA, 3Mayo Clinic, Rochester, MN Purpose/Objective(s): Homologous recombination (HR) is involved in the repair of DNA double strand breaks (DSBs), particularly in
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International Journal of Radiation Oncology Biology Physics
proliferating tumor cells. Hereditary breast cancer patients with BRCA1 or BRCA2 loss of function and impaired HR potentially benefit from DNA repair targeted therapies including poly-ADP ribose polymerase (PARP) inhibitors. Our previous work has suggested that sporadic breast cancers without a BRCA1/2 mutation can also show HR deficiency. Therefore, we extended this work by screening breast cancer cell lines for reduced HR to determine the underlying cause of the functional defect. Materials/Methods: Cell lines were screened for HR function by exposing to ionizing radiation (IR) and assessing for protein recruitment to nuclear foci using immunofluorescence microscopy. Lines found to be defective for HR were verified by the DR-GFP assay of HR and sensitivity to mitomycin C (MMC) and PARP inhibitors using colony survival assays. In addition, array comparative genomic hybridization (aCGH) was utilized to examine global genomic instability of the HR defective cell lines. Genetic or protein alterations from any part of the BRCA1-BRCA2 pathway were determined. Results: In comparison to HR proficient cell lines, which showed a robust induction of RAD51 foci after 10 Gy, several cell lines demonstrated defective HR by a lack of induced RAD51 foci. The DR-GFP assay confirmed the HR deficiency suggested by reduced RAD51 foci. All cell lines showed induction of the established upstream DNA damage response (DDR) proteins from Y-H2AX to RAP80. However, a disruption of HR at the level of Abraxas, a protein in the DDR pathway that is involved in BRCA1 recruitment to DNA damage sites, was found. None of the HRdefective cell lines have BRCA1 or BRCA2 mutations. Abnormalities in post-translational modifications of BRCA1 pathway proteins are under investigation. The response of cells to MMC was usually sensitive, with some variability. One cell line, shown to be deficient in HR, still proved to be relatively resistant to MMC treatment, suggesting that cell survival is a more complex end-point. We are investigating the underlying mechanism of crosslink resistance in this cell line, specifically querying whether RAD52 is mediating the resistance in the absence of BRCA1 recruitment. Conclusions: The normal variation in patient tumor phenotypes is reflected in the responses found in sporadic breast cancer cell lines. Defects in BRCA1 recruitment are found, usually resulting in sensitivity to crosslinking agents. Our research may help identify a potentially larger pool of patients able to benefit from DNA repair targeted therapies. Author Disclosure: M. Thompson: None. R. Mutter: None. S. Powell: None.
damage by CLDR. The significantly decreased Bcl-2/Bax ratio induced by CLDR may contribute to the increased cell death of A549. Thus, the improved inhibition of CLDR on lung cancer cell growth may be mediated, at least partially, by the decrease Bcl2/Bax ratio and cyclin B1-related G2/M arrest. Author Disclosure: A. Qu: None. W. Hao: None. L. Jinna: None. W. Junjie: None. L. Jingjia: None. H. Yuzhu: None. H. Li: None. Z. Yong: None.
3137 Direct Effects of 125I Seeds Irradiation on A549 Lung Cancer Cells: G2/M Arrest and Enhanced Cell Death A. Qu,1 W. Hao,1 L. Jinna,1 W. Junjie,1 L. Jingjia,1 H. Yuzhu,2 H. Li,1 and Z. Yong2; 1Peking University Third Hospital, Beijing, China, 2Institute of Zoology, Chinese Academy of Sciences, Beijing, China Purpose/Objective(s): External beam radiation (EBRT) and 125I seed continuous low dose rate radiation (CLDR) are clinically used to treat lung cancers. The reasons for the improved therapy of CLDR were not fully understood. We herein investigated the different direct effects of EBRT and CLDR on lung cancer cells and its related mechanisms. Materials/Methods: A549 cells were exposed to different doses of EBRT and CLDR, respectively. A 6 MV-X ray linear accelerator was used for EBRT and 125I seeds were used for CLDR. Cell survival, death, cycle and apoptosis were detected. The related intracellular molecule alterations were determined by real-time PCR and Western blotting. Results: Significantly lower colony forming efficiency of A549 cells was observed after CLDR than EBRT. CLDR inhibited cell growth and induced apoptosis, G2/M arrest more efficiently EBRT. Cyclin B1 levels were reduced by CLDR. p-H2AX (Ser139) and DNA-PKcs expression were significantly elevated in A549 cells after CLDR. anti-apoptosis Bcl-2 protein levels in A549 cells were significantly reduced and apoptosisinducing Bax mRNA and protein levels were increased after CLDR. Conclusions: CLDR was more efficient to inhibit A549 lung cancer cell growth than EBRT. Decreased cyclin B1 may mediate CLDR-induced G2/ M arrest. The long-lasting p-H2AX activity indicates the enhanced DNA
3138 Radiation Therapy-Related Changes in Serum Proteome Patterns Are Affected by Medium Doses of Radiation Delivered to Large Volumes of Normal Tissue P. Widlak,1 M. Pietrowska,1 J. Polanska,2 T. Rutkowski,1 A. Wygoda,1 R. Tarnawski,1 and K. Skladowski1; 1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland, 2Institute of Automatics Control, Silesian University of Technology, Gliwice, Poland Purpose/Objective(s): Intensity-modulated radiation therapy (IMRT) involves irradiation of increased volume of normal tissue with low and medium doses, which biological relevance is not clear yet. In current work the association of early biological effects of RT with dose and volume of irradiated tissue was analyzed. Serum proteome features were used as a surrogate for general reaction of whole patient’s organism while acute mucosal reaction reflected local effect of irradiation. Materials/Methods: Seventy patients with head and neck squamous cell cancer received definitive treatment with accelerated regimen (CAIR). Blood samples were collected before RT, after two weeks of RT and one month after the end of RT; acute mucosal reactions were registered during the treatment. The low-molecular-weight fraction of serum proteome was analyzed using MALDI-ToF mass spectrometry in the 800-14,000 Da range. Correlations between therapy-induced changes in abundances of specific serum components and parameters reflecting absorbed doses of radiation were analyzed. Results: The major changes in serum proteome profiles were observed between samples collected before and on completion of RT. RT-related changes in serum proteome features were associated with total radiation energy absorbed by patient’s body and resulted primarily from low/ medium doses delivered to large fraction of body mass. Changes of specific serum proteome features also correlated with the maximal intensity of acute mucosal reaction (AMR). Noteworthy, the intensity of AMR apparently correlated with medium doses of radiation absorbed by large volumes of normal tissue. Conclusions: This is the first time when effects of low/medium doses of radiation delivered to large fraction of normal tissue were documented at the level of serum proteome, which apparently reflected general response of patient’s organism. This finding, together with observed correlation between the AMR intensity and irradiation of normal tissues with medium doses of radiation, may have clinical implication. Author Disclosure: P. Widlak: None. M. Pietrowska: None. J. Polanska: None. T. Rutkowski: None. A. Wygoda: None. R. Tarnawski: None. K. Skladowski: None.
3139 Investigation of Stem-Like Cells Role in Regional Radiosensitivity of the Lung O. Maria,1 A. Maria,2 N. Ybarra,1 K. Jeyaseelan,1 S. Lee,1 J. Perez,1 S.M. Lehnert,1 M. Serban,1 J. Seuntjens,1 and I. El Naqa1; 1McGill University, Montreal, QC, Canada, 2Tanta University, Tanta, Egypt Purpose/Objective(s): The goal of this study is to investigate the difference in stem-like cells distribution between the upper and the lower lobes of the lung and their potential role in explaining variability in regional radiosensitivity of normal lung tissue and incidences of radiation-induced lung damage. Materials/Methods: Fourteen male Sprague-Dawley rats (8 weeks, 200250 g) were grouped into two main groups: control (sham radiation, n Z 5) and treatment (irradiated, n Z 9) groups. The treatment group received
3134 A Mitotic Pathway for Radiation-Induced Genome Damage S. Bakhoum,1,2 L. Kabeche,1 M. Wood,3 A. Suriawinata,1 R. Louie,3 D. Chan,3 C. Petritsch,3 J. Murnane,3 D. Compton,1 and B. Zaki1,2; 1Geisel School of Medicine at Dartmouth, Hanover, NH, 2Norris-Cotton Cancer Center, Lebanon, NH, 3University of California San Francisco, San Francisco, CA Purpose/Objective(s): The exquisite sensitivity of mitotic cancer cells to ionizing radiation underlies the rationale for fractionated radiation therapy. Nonetheless, the mechanism of this cell cycle-dependent vulnerability is unknown. Materials/Methods: Here we use live cell imaging and high-resolution microscopy to examine the effect of radiation on chromosome segregation and genome damage during mitosis. We also investigate the role of chromosome segregation errors on the viability of irradiated cells using clonogenic survival assays, an orthotopic mouse model of glioblastoma multiforme, and a retrospective clinical study in patients diagnosed with rectal adenocarcinoma. Results: We show that ionizing radiation selectively increases the stability of microtubule attachments to chromosomes at kinetochores, eliciting a dose-dependent surge of kinetochore-microtubule attachment errors during chromosome segregation. These errors, manifested by lagging chromosomes in anaphase, generate whole-chromosome mis-segregation, long-term aneuploidy, a preponderance of micronuclei, and chromosome pulverization leading to chromothripsis. Destabilizing microtubule attachments to chromosomes leads to reduction of these defects, substantial increase in the viability of irradiated mitotic cells (by up to 500-fold), and radiation resistance in orthotopically transplanted human glioblastoma multiforme tumors. Yet, pharmacologically increasing kinetochore-microtubule attachment errors potentiates radiation-induced genome damage. Consistently, we also show that rates of lagging chromosomes predict response to chemoradiation therapy in patients with
Poster Viewing Abstracts S637 rectal adenocarcinoma whereby patients with elevated chromosome missegregation rates exhibit a superior pathological response (OR Z 4.21). Conclusions: Collectively, our data identify a novel mitotic pathway for widespread genome damage beyond direct DNA breaks, and they suggest that kinetochore-microtubules can be usurped to generate this damage for the purpose of tumor radiosensitization. Author Disclosure: S. Bakhoum: None. L. Kabeche: None. M. Wood: None. A. Suriawinata: None. R. Louie: None. D. Chan: None. C. Petritsch: None. J. Murnane: None. D. Compton: None. B. Zaki: None.
3135 HIF-1alpha Posttranscriptional mRNA Regulation Under Normoxia: The Significance of UTRs T. Nishioka, M. Yasuda, and H. Shirato; Hokkaido University, Sapporo, Japan Purpose/Objective(s): HIF-1alpha is a central regulator of cancer progression. HIF-1alpha protein is rapidly degraded at normal oxygen level. Under hypoxia, it escapes from degradation and reaches to a detectable level. In the present study, we investigated: 1) the relation between HIF-1alpha and radio-sensitivity, 2) whether or not HIF-1alpha degradation is due to VHL, and 3) the effect of HIF-1alpha’s 5’- and 3’UTR on translation. Materials/Methods: 1) Colony assay was performed on normal rat fibroblasts that were transfected with HIF-1alpha at a dose 4, 8, and 10 Gy. 2) VHL: Western blotting was carried out on several malignant cell lines expressing different amount of endogenous HIF-1alpha. 3) Luciferase reporter assay: a series of HIF-1alpha constructs containing coding region (C) with or without UTRs were made and introduced into HeLa, H1299, HEK293, B16 F0, B16F10, U87MG, PPC-C2A, MCF7, and MDA-MB453 to measure HRE-Luciferase activity in each cell line. RT-PCR and immunoblotting were also performed at the same experimental conditions. Results: 1) Cells transfected with HIF-1alpha expressed significantly higher survival at each dose (p < 0.05). 2) The VHL level did not always correspond with the expression of HIF-1alpha. 3) For each cell line, deletion of 5’UTR showed a decreased HIF-1alpha expression and deletion of 3’UTR enhanced the expression. A synergetic effect of more than 2-fold was observed for deletion (i.e., 5’ and 3’) for each cell lines. In particular, a highly-aggressive breast cancer cell line MDA-MB-453 demonstrated a 3.8-fold higher transactivational activity compared to a less-aggressive breast cancer cell line MCF7. As to the deletion 5’UTR mutants, a 15- to 20-fold increased transactivational activity was observed for 5’UTR containing constructs compared to the coding region only. RT-PCR revealed that all of HIF-1 alpha constructs expressed the same amount of mRNA. Surprisingly, the cells transfected with 5’UTR containing HIF-1alpha expressed 10 times more amount of proteins compared with coding region only clone. Conclusions: 1) HIF-1alpha increases radio-resistance. 2) In some cells, higher level of HIF-1alpha expression is not due to the expression level of VHL. 3) HIF-1alpha UTRs affects its expression. HIF-1alpha 5’ UTR has an effect of increased protein expression while 3’UTR reduces the protein level. The extent of UTR effect on translation might explain aggressive nature as seen in the breast cancer cell lines. If detailed mechanisms of UTRs on HIF-1alpha translation are clarified, a new path is expected to radiation therapy. Author Disclosure: T. Nishioka: None. M. Yasuda: None. H. Shirato: None.
3136 Screening for BRCA1-pathway Inactivation in Sporadic Breast Cancer Cell Lines M. Thompson,1,2 R. Mutter,3 and S. Powell1; 1Memorial Sloan-Kettering Cancer Center, New York, NY, 2University of Pittsburgh School of Medicine, Pittsburgh, PA, 3Mayo Clinic, Rochester, MN Purpose/Objective(s): Homologous recombination (HR) is involved in the repair of DNA double strand breaks (DSBs), particularly in
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International Journal of Radiation Oncology Biology Physics
proliferating tumor cells. Hereditary breast cancer patients with BRCA1 or BRCA2 loss of function and impaired HR potentially benefit from DNA repair targeted therapies including poly-ADP ribose polymerase (PARP) inhibitors. Our previous work has suggested that sporadic breast cancers without a BRCA1/2 mutation can also show HR deficiency. Therefore, we extended this work by screening breast cancer cell lines for reduced HR to determine the underlying cause of the functional defect. Materials/Methods: Cell lines were screened for HR function by exposing to ionizing radiation (IR) and assessing for protein recruitment to nuclear foci using immunofluorescence microscopy. Lines found to be defective for HR were verified by the DR-GFP assay of HR and sensitivity to mitomycin C (MMC) and PARP inhibitors using colony survival assays. In addition, array comparative genomic hybridization (aCGH) was utilized to examine global genomic instability of the HR defective cell lines. Genetic or protein alterations from any part of the BRCA1-BRCA2 pathway were determined. Results: In comparison to HR proficient cell lines, which showed a robust induction of RAD51 foci after 10 Gy, several cell lines demonstrated defective HR by a lack of induced RAD51 foci. The DR-GFP assay confirmed the HR deficiency suggested by reduced RAD51 foci. All cell lines showed induction of the established upstream DNA damage response (DDR) proteins from Y-H2AX to RAP80. However, a disruption of HR at the level of Abraxas, a protein in the DDR pathway that is involved in BRCA1 recruitment to DNA damage sites, was found. None of the HRdefective cell lines have BRCA1 or BRCA2 mutations. Abnormalities in post-translational modifications of BRCA1 pathway proteins are under investigation. The response of cells to MMC was usually sensitive, with some variability. One cell line, shown to be deficient in HR, still proved to be relatively resistant to MMC treatment, suggesting that cell survival is a more complex end-point. We are investigating the underlying mechanism of crosslink resistance in this cell line, specifically querying whether RAD52 is mediating the resistance in the absence of BRCA1 recruitment. Conclusions: The normal variation in patient tumor phenotypes is reflected in the responses found in sporadic breast cancer cell lines. Defects in BRCA1 recruitment are found, usually resulting in sensitivity to crosslinking agents. Our research may help identify a potentially larger pool of patients able to benefit from DNA repair targeted therapies. Author Disclosure: M. Thompson: None. R. Mutter: None. S. Powell: None.
damage by CLDR. The significantly decreased Bcl-2/Bax ratio induced by CLDR may contribute to the increased cell death of A549. Thus, the improved inhibition of CLDR on lung cancer cell growth may be mediated, at least partially, by the decrease Bcl2/Bax ratio and cyclin B1-related G2/M arrest. Author Disclosure: A. Qu: None. W. Hao: None. L. Jinna: None. W. Junjie: None. L. Jingjia: None. H. Yuzhu: None. H. Li: None. Z. Yong: None.
3137 Direct Effects of 125I Seeds Irradiation on A549 Lung Cancer Cells: G2/M Arrest and Enhanced Cell Death A. Qu,1 W. Hao,1 L. Jinna,1 W. Junjie,1 L. Jingjia,1 H. Yuzhu,2 H. Li,1 and Z. Yong2; 1Peking University Third Hospital, Beijing, China, 2Institute of Zoology, Chinese Academy of Sciences, Beijing, China Purpose/Objective(s): External beam radiation (EBRT) and 125I seed continuous low dose rate radiation (CLDR) are clinically used to treat lung cancers. The reasons for the improved therapy of CLDR were not fully understood. We herein investigated the different direct effects of EBRT and CLDR on lung cancer cells and its related mechanisms. Materials/Methods: A549 cells were exposed to different doses of EBRT and CLDR, respectively. A 6 MV-X ray linear accelerator was used for EBRT and 125I seeds were used for CLDR. Cell survival, death, cycle and apoptosis were detected. The related intracellular molecule alterations were determined by real-time PCR and Western blotting. Results: Significantly lower colony forming efficiency of A549 cells was observed after CLDR than EBRT. CLDR inhibited cell growth and induced apoptosis, G2/M arrest more efficiently EBRT. Cyclin B1 levels were reduced by CLDR. p-H2AX (Ser139) and DNA-PKcs expression were significantly elevated in A549 cells after CLDR. anti-apoptosis Bcl-2 protein levels in A549 cells were significantly reduced and apoptosisinducing Bax mRNA and protein levels were increased after CLDR. Conclusions: CLDR was more efficient to inhibit A549 lung cancer cell growth than EBRT. Decreased cyclin B1 may mediate CLDR-induced G2/ M arrest. The long-lasting p-H2AX activity indicates the enhanced DNA
3138 Radiation Therapy-Related Changes in Serum Proteome Patterns Are Affected by Medium Doses of Radiation Delivered to Large Volumes of Normal Tissue P. Widlak,1 M. Pietrowska,1 J. Polanska,2 T. Rutkowski,1 A. Wygoda,1 R. Tarnawski,1 and K. Skladowski1; 1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland, 2Institute of Automatics Control, Silesian University of Technology, Gliwice, Poland Purpose/Objective(s): Intensity-modulated radiation therapy (IMRT) involves irradiation of increased volume of normal tissue with low and medium doses, which biological relevance is not clear yet. In current work the association of early biological effects of RT with dose and volume of irradiated tissue was analyzed. Serum proteome features were used as a surrogate for general reaction of whole patient’s organism while acute mucosal reaction reflected local effect of irradiation. Materials/Methods: Seventy patients with head and neck squamous cell cancer received definitive treatment with accelerated regimen (CAIR). Blood samples were collected before RT, after two weeks of RT and one month after the end of RT; acute mucosal reactions were registered during the treatment. The low-molecular-weight fraction of serum proteome was analyzed using MALDI-ToF mass spectrometry in the 800-14,000 Da range. Correlations between therapy-induced changes in abundances of specific serum components and parameters reflecting absorbed doses of radiation were analyzed. Results: The major changes in serum proteome profiles were observed between samples collected before and on completion of RT. RT-related changes in serum proteome features were associated with total radiation energy absorbed by patient’s body and resulted primarily from low/ medium doses delivered to large fraction of body mass. Changes of specific serum proteome features also correlated with the maximal intensity of acute mucosal reaction (AMR). Noteworthy, the intensity of AMR apparently correlated with medium doses of radiation absorbed by large volumes of normal tissue. Conclusions: This is the first time when effects of low/medium doses of radiation delivered to large fraction of normal tissue were documented at the level of serum proteome, which apparently reflected general response of patient’s organism. This finding, together with observed correlation between the AMR intensity and irradiation of normal tissues with medium doses of radiation, may have clinical implication. Author Disclosure: P. Widlak: None. M. Pietrowska: None. J. Polanska: None. T. Rutkowski: None. A. Wygoda: None. R. Tarnawski: None. K. Skladowski: None.
3139 Investigation of Stem-Like Cells Role in Regional Radiosensitivity of the Lung O. Maria,1 A. Maria,2 N. Ybarra,1 K. Jeyaseelan,1 S. Lee,1 J. Perez,1 S.M. Lehnert,1 M. Serban,1 J. Seuntjens,1 and I. El Naqa1; 1McGill University, Montreal, QC, Canada, 2Tanta University, Tanta, Egypt Purpose/Objective(s): The goal of this study is to investigate the difference in stem-like cells distribution between the upper and the lower lobes of the lung and their potential role in explaining variability in regional radiosensitivity of normal lung tissue and incidences of radiation-induced lung damage. Materials/Methods: Fourteen male Sprague-Dawley rats (8 weeks, 200250 g) were grouped into two main groups: control (sham radiation, n Z 5) and treatment (irradiated, n Z 9) groups. The treatment group received